JP7446238B2 - Devices and uses thereof for maintaining metal homeostasis - Google Patents

Description

The present invention relates to the field of medical devices, and more particularly to devices for extracting metals from the body. These devices can be used, for example, for the prevention and/or treatment of pathologies associated with dysregulation of metal homeostasis in the body, such as neurological diseases.

The potentially harmful effects of metals in the body, especially at the neurological level, are highlighted by a growing number of scientific studies (C. Marchetti et al., Biometals, 2014). Chelation therapy aimed at reducing the concentration of metal ions has already been used for many years in cases of acute metal poisoning. Several chelating agents, each related to a specific group of metals, are already tolerated in humans (G. Crisponi et al., Coordination Chemistry Reviews, 2015). Chelation therapy has also been shown to be an essential tool in the treatment of transfused patients with β-thalassemia. Patients who have received multiple blood transfusions suffer from iron buildup in their bodies. These iron deposits have been regulated by intravenous or oral administration of iron chelators such as desferoxamine, deferiprone, or deferasirox (PV Bernhardt et al., Dalton Trans, 2007). Chelation therapy with D-penicillamine and trientine (orally) is also currently used to extract copper cations and treat Wilson's disease, which is caused by a genetic abnormality affecting the copper transporter ATP7B. This abnormality results in copper overload, increasing circulating copper in the blood and resulting in deposits in organs, primarily the liver and brain (M.L. Schilsky, Clin. Liver. Dis., 2017). Chelation therapy has good efficacy in the case of pre-symptomatic treatment, but less efficacy in the case of liver or nervous system damage (M. Wiggelinkhuizen et al., Aliment Pharmacol., 2009). This is likely due to the difficulty of reaching the target area and the low specificity.

Unfortunately, chelation therapy is also being misused intravenously to treat many other conditions (autism, intermittent claudication, etc.) without prior medical validation. Their misuse can lead to serious side effects and even death of the patient in the most tragic cases due to excessive dysregulation of essential metal homeostasis in the body (G. Crisponi et al., Coordination Chemistry Reviews , 2015).

A growing body of scientific research highlights the important role that metals, particularly iron, but also copper, zinc, manganese, and even aluminum, play in many neurological disorders (E. J. McAllum et al., J. Mol. Neurosci., 2016). This is a necessary example of a neurological disorder due to iron overload, a rare disease associated with genetic abnormalities associated with iron accumulation in certain areas of the brain, which currently only benefits from palliative treatment. (S. Wiethoff et al., Handb. Clin. Neurol., 2017). Furthermore, many studies have shown that iron tends to accumulate in the brain with age (J. Acosta-Cabronero et al., Journal of Neuroscience, 2016). Iron plays an important role in many brain functions such as mitochondrial respiration, myelin and neurotransmitter synthesis, and metabolism (AA Belaidi et al., Journal of Neurochemistry, 2016). In the brain, iron is localized primarily in the substantia nigra pars compacta and central gray nucleus at levels similar to those in the liver. Iron tends to accumulate in certain areas of the brain with age, where it is primarily found associated with ferritin and neuromelanin. The areas where iron levels are most likely to increase are the substantia nigra, putamen, globus pallidus, caudate, or cortex, each of which is associated with different neurodegenerative disorders (D.J. Hare et al., Nat. Rev. Neurol., 2015). Several neurological diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, are accompanied by elevated levels of iron in specific areas, leading to cellular damage and oxidative stress (AA Belaidi et al. Journal of Neurochemistry, 2016). In Parkinson's disease, increased amounts of iron have been observed in the substantia nigra, an area of the brain susceptible to parkinsonian pathogenesis. Elevated iron levels are specific to the substantia nigra and do not occur in other areas not affected by the disease. This increased level of iron can lead to damage following the Fenton reaction, and it has been established that oxidative damage is one of the hallmarks of neurodegenerative diseases (S. Ayton et al., Biomed. Res. Int. , 2014). Alzheimer's disease is also characterized by disturbances in the amount of metals in the brain, but also involves other areas of the brain and other proteins. Indeed, in this case an increase in the level of iron and a decrease in the level of copper seem to be observed (S.F. Graham et al., J. Alzheimers Dis., 2014). Huntington's disease is another neurodegenerative disease that includes movement disorders, cognitive decline, and psychotic challenges. In this lesion, many markers of oxidative stress are observed in the brain, which may be related to dysregulation of iron homeostasis (S.J.A. van den Bogaard et al., International Review of Neurobiology , 2013). Elevated levels of iron in several brain regions (putamen, caudate nucleus, and globus pallidus) have been shown in several studies, including the work of Bartzorkis and colleagues (G. Bartzorkis et al., Archives of Neurology, 1999). It has been verified by MRI studies.

Abundant evidence regarding the role of iron homeostasis dysregulation in many neurodegenerative disorders has led scientists to study the effects of chelation therapy on these pathologies (Table 1). For example, deferiprone (used for the treatment of iron deposits that occur during blood transfusions for β-thalassemia) was used in a phase II clinical trial (DeferipronPD, NCT01539837) that included 22 patients (A. Martin- Bastida et al., Scientific Reports, 2017). In this clinical trial, treatment continued for 6 months and was well tolerated by patients. Decreased iron levels were observed in the dentate and caudate nuclei. A reduction in iron levels in the substantia nigra was observed in only 3 patients. No changes in iron levels in the globus pallidus and putamen were observed. The trial showed a trend toward improvement in motor scores and quality of life, but it was not statistically significant. Another trial with deferiprone was conducted by another team (Fair-Park I), which showed a reduction in iron levels in the substantia nigra and an improvement in motor scores, but this also did not reach statistical significance. (G. Grolez et al., BMC Neurology, 2015). As the results of the Fair-Park I trial were encouraging, the Fair-Park II trial was initiated in 2016 (Table 1). In view of the encouraging results of trials in Parkinson's disease, deferiprone was recently proposed for clinical trials in Alzheimer's disease (Table 1). Another metal chelator, clioquinol, had already been tested to study its effect on amyloid fibril formation (Table 1). This drug was banned in the 1970s due to a suspected association with myelooptic neuropathy (C.W. Ritchie et al., Arch. Neurol., 2003) and was reevaluated in this study. Although some adverse effects were reported in this study, the safety of this product is considered sufficient for future clinical trials and clinical benefit is expected in patients most susceptible to this disease. observed. Following this trial, a clioquinol derivative (PBT2) was developed and entered Phase IIa trials (L. Lannfelt et al., Lancet Neurol., 2008). Good tolerance to treatment was observed. Decreased levels of Aβ protein were observed in the cerebrospinal fluid, but not in the plasma. Two executive functions also improved in treated patients.

Thus, several iron chelators such as desferrioxamine, clioquinol, MAO, Vk-28, M30, or M30A (N. Wang et al., Biomacromolecules, 2017) have been proposed for chelation treatment of neurodegenerative diseases. has attracted the attention of researchers during preclinical or even clinical trials. Nevertheless, the effectiveness of these molecules and other iron chelators is limited by their short lifetime in the body, potential for cytotoxicity at high doses, and ability to cross the blood-brain barrier into the brain. There are still limitations due to difficulty in targeting the most affected areas of the body, as well as pre-saturation with endogenous cations.

In parallel to these studies, clinical trials were requested in 2001 by the National Institutes of Health (NIH) to confirm the benefits of EDTA for the treatment of cardiovascular disease using rigorous scientific protocols. . Indeed, it was claimed in the 1950s that EDTA could chelate calcium in atherosclerotic plaques and cause its breakdown. Most cardiologists had rejected this implementation because of the lack of clinical results. Nevertheless, clinicians continued to use it, and in 2007, studies showed that more than 110,000 patients were receiving the treatment annually in the United States. In 2002, NIH funded the Trial to Assess Chelation Therapy (TACT, NCT, 00044213). The study previously enrolled 1,708 patients aged 50 and older who had suffered a myocardial infarction for at least six months. The trial showed good tolerance to EDTA therapy in enrolled patients (DB. Mark et al., Circ. Cardiovasc. Qual. Outcomes, 2014). A modest but significant effect was observed for patients treated with EDTA (P. Ouyang et al., Curr. Cardiol. Rep., 2015). However, this effect was much greater in the subgroup of patients with diabetes (633 patients) (E. Escolar et al., Circ. Cardiovasc. Qual. Outcomes, 2014). That is, diabetic patients treated with EDTA had a 41% reduction in relative risk (p<0.001) for combined cardiovascular metrics and a 40% reduction in risk of nonfatal stroke or nonfatal myocardial infarction. (p=0.017) and a 43% (p=0.011) reduction in mortality risk. Thus, this study shows the potential of targeted chelation therapy to avoid future attacks for a specific group of patients, namely those suffering from diabetes.

Currently, it has recently been shown to be associated with symptoms of cocaine dependence (K.D. Ersche et al., Transl. Psychiatry, 2017), and is suspected to be associated with syndromes such as autism (D.A. Rossignol et al., Transl. Psychiatry, 2017). It has also been discussed that an increasing number of pathologies are associated with dysregulation of metal homeostasis in the body, such as (2014). Although many publications have emphasized the role of iron visualized by MRI,
(i) Manganese in the so-called manganese toxicity neurological syndrome (P. Chen et al., J. Neurochem., 2015),
(ii) other endogenous metals such as copper in cases of Wilson's disease, or (i) mercury for neurotoxicity and cardiac damage (J. Ohlander et al., Int. J. Occup. Environ. Health, 2016);
(ii) cadmium, for example in cases of poisoning (VM Andrade, Adv. Neurobiol, 2017);
(iii) Lead associated with lead poisoning (G. Bjorklund et al., Arch. Toxicol., 2017)
Exogenous metals have also been shown to cause severe neurological disorders when their homeostasis is deregulated by external factors or genetic abnormalities.

C. Marchetti et al., Biometals, 2014 G. Crisponi et al., Coordination Chemistry Reviews, 2015 P. V. Bernhardt et al., Dalton Trans, 2007 M. L. Schilsky, Clin. Liver. Dis. , 2017 M. Wiggelinkhuizen et al., Aliment Pharmacol. , 2009 E. J. McAllum et al., J. Mol. Neurosci. , 2016 S. Wiethoff et al., Handb. Clin. Neurol. , 2017 J. Acosta-Cabronero et al., Journal of Neuroscience, 2016 A. A. Beladi et al., Journal of Neurochemistry, 2016 D. J. Hare et al., Nat. Rev. Neurol. , 2015 S. Ayton et al., Biomed. Res. Int. , 2014 S. F. Graham et al., J. Alzheimers Dis. , 2014 S. J. A. van den Bogaard et al., International Review of Neurobiology, 2013 G. Bartzorkis et al., Archives of Neurology, 1999 A. Martin-Bastida et al., Scientific Reports, 2017 G. Grolez et al., BMC Neurology, 2015 C. W. Ritchie et al., Arch. Neurol. , 2003 L. Lannfelt et al., Lancet Neurol. , 2008 N. Wang et al., Biomacromolecules, 2017 D. B. Mark et al., Circ. Cardiovasc. Qual. Outcomes, 2014 P. Ouyang et al., Curr. Cardiol. Rep. , 2015 E. Escolar et al., Circ. Cardiovasc. Qual. Outcomes, 2014 K. D. Ersche et al., Transl. Psychiatry, 2017 D. A. Rossignol et al., Transl Psychiatry, 2014 P. Chen et al., J. Neurochem. , 2015 J. Ohlander et al., Int. J. Occup. Environ. Health, 2016 V. M. Andrade, Adv. Neurobiol, 2017 G. Bjorklund et al., Arch. Toxicol. , 2017 C. M. Kho, Mol. Neurobiol. , 2016

There is therefore a need today to develop new means of extracting metals from the body with the aim of preventing and/or treating pathologies associated with dysregulation of metal homeostasis. These will provide one or more of the following advantages.
- Targeting and extracting metals from the body, regardless of whether they are present in large amounts or in small amounts.
- Regulate homeostasis of essential metals.
- No cytotoxicity.
- There is no limit to its lifespan in the body.
- Promote action across the blood-brain barrier in the treatment of neurological diseases.
- It has applications that can be adapted to the prevention and/or treatment of any pathologies associated with the dysregulation of metal homeostasis.

These and other advantages are described in the disclosure below.

Therefore, in this context, the inventors of the present invention have developed a medical device comprising at least one chelating agent for extracting metal cations.

In a first aspect, the invention relates to a device for maintaining metal homeostasis for therapeutic purposes, characterized in that it comprises means for extracting metal cations.

"Maintaining metal homeostasis for therapeutic purposes" means regulating the levels of certain metals in the body, especially with the purpose of extracting excess metal cations that may be involved in pathologies.

In one embodiment, the term "metal homeostasis" refers to the homeostasis of a metal cation (more specifically, the homeostasis of a particular metal cation).

In one embodiment, said means for extracting metal cations comprises:
- an implant to which at least one chelating agent is grafted, or - an irrigation fluid comprising at least one chelating agent.

According to the invention, the term "chelating agent" means an organic group capable of complexing with at least one metal cation. According to a preferred embodiment, the chelating agent is capable of complexing with the metal cations that it is desired to extract, and the complexation constant log(K _C1 ) of said chelating agent with at least one of said metal cations is greater than 10, in particular 11, 12, 13, 14, 15, preferably greater than or equal to 15. Advantageously, the chelating agent is a metal such as copper (Cu), iron (Fe), zinc (Zn), mercury (Hg), cadmium (Cd), lead (Pb), aluminum (Al), manganese (Mn), Arsenic (As), mercury (Hg), cobalt (Co), nickel (Ni), vanadium (V), tungsten (W), zirconium (Zr), titanium (Ti), chromium (Cr), silver (Ag), Bismuth (Bi), tin (Sn), selenium (Se), thallium (Th), calcium (Ca), magnesium (Mg), scandium (Sc), yttrium (Y), lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), Thulium (Tm), Ytterbium (Yb), Lutetium (Lu), Actinium (Ac), Thorium (Th), Protactinium (Pa), Uranium (U), Neptunium (Np), Plutonium (Pu), Americium (Am) , curium (Cm), berkelium (Bk), californium (Cf), einsteinium (Es), fermium (Fm), mendelevium (Md), nobelium (No), and lawrencium (Lr). It becomes a complex. Even more advantageously, the chelating agent is complexed with at least one of the cations of the metals copper, iron, zinc, mercury, cadmium, lead, aluminium, manganese, magnesium, calcium, and gadolinium, especially with manganese and gadolinium. do. Even more advantageously, the chelating agent is complexed with at least one of the metal copper, iron and/or zinc cations.

According to the invention, the term "at least one chelating agent" means a single type of chelating agent, a mixture of different chelating agents or a mixture of several identical chelating agents.

Advantageously, the specificity of the chelating agent for said metal to be extracted (metal cation) is high compared to other cationic trace elements, in particular the difference in complexation constants is preferably greater than 3, more particularly The difference in complexing constants between calcium and magnesium is preferably greater than 3, and even greater than 5.

According to a preferred embodiment, the device also comprises trace elements selected from calcium, magnesium, iron, copper, zinc and manganese, either directly in the polymer, implant or solid, or in the perfusate. This makes it possible, for example, to regulate the homeostasis of essential metals.

According to a preferred embodiment, said means of said device are adapted to remove biological fluids, organs or tissues from biological fluids, organs or tissues, in particular when the content of metal cations is less than 1 ppm, in particular 0.1 ppm, 0.01 ppm, preferably less than 1 ppb. making it possible to extract the metal cations. Advantageously, at least more than half of the cations present can be extracted.

According to the present invention, the term "biological fluid" means any fluid, such as blood, cerebrospinal fluid, synovial fluid, or ascites fluid, with which the device of the present invention is contacted.

According to the present invention, the term "organ" includes any organ, such as the brain, liver, pancreas, intestines, or lungs, which can be contacted with or into which a device of the present invention can be implanted or inserted. means an organ.

According to the present invention, the term "tissue" includes any tissue (where applicable means tumor tissue). For example, the device is contacted, inserted or implanted endoscopically, especially into a tumor.

According to a preferred embodiment, said means for extracting metal cations is, for example, a material, intended to extract metal cations in an amount corresponding to at least 1% of its mass, preferably more than 10% of its mass. enable.

The means for metal extraction is a dialysis system.
Advantageously and according to a preferred embodiment, the means for extracting metal cations are
a. a porous dialysis membrane, and b. A dialysis system that includes a reservoir containing perfusate.

According to the invention, the term "dialysis system" is any system that passes metal cations through an artificial membrane.

According to this particular embodiment, said device is advantageously a microdialysis system. Over the years, new techniques for local analyte or sample collection or local drug delivery (microdialysis) have been developed. Microdialysis was developed at the end of the 1950s to collect and deliver various substances in areas of interest (CM Kho, Mol. Neurobiol., 2016). Microdialysis makes it possible to collect or deliver only samples that can pass through a semipermeable membrane whose cut-off threshold is chosen according to the intended use. In the case of dialysis, this is often a dynamic diffusion phenomenon driven by differences in the concentration of the diffusing species between each side of the membrane. For low concentrations of species, the driving force often reaches a limit or saturates quickly, and capture of the species of interest is limited by the equilibrium concentration.

Advantageously, the microdialysis device according to the invention bypasses the problems of conventional chelators by maintaining at least one target metal-complexing species inside the dialysis membrane, and at extremely high rates. Enables local extraction of target metal ions. The complexing species is present in a polymer or nanoparticle with a mass greater than the membrane cutoff, and therefore the complexing species remains in the fluid within the dialysis membrane (i.e., the perfusate). There is. A dialysis device containing the complexing species is then placed in the desired area, such as the brain for the treatment of neurodegenerative diseases.

Cations smaller than the membrane cutoff are able to diffuse through the membrane into the solution containing the chelating agent. The strong complexing properties of the ligands used make it possible to chelate the target metal even when the target metal is present in very small amounts. Therefore, this chelation reduces the concentration of free target ions in solution inside the membrane, maintains a strong concentration gradient of target metal ions between the concentrations outside and inside the membrane, increases the extraction time, and increases the concentration of cations. flow is maintained. Equivalent concentrations of these ions may be placed in the dialysis membrane in order not to interfere with the homeostasis of other metal cations.

According to the invention, any microdialysis device known to those skilled in the art may be used, as long as it comprises a porous dialysis membrane and a reservoir containing a perfusate comprising at least one chelating agent as described above. In this regard, the cut-off threshold of the porous membrane is lower than the mass of the chelating agent. By way of example, devices that can be used in connection with the present invention include medical devices developed by M Dialysis AB (Sweden), such as microdialysis catheters (product numbers 8010509, P000049, 8010337; this list is not exhaustive) There is.

According to this preferred embodiment, the perfusate is a colloidal suspension of nanoparticles whose average diameter is larger than the pores of said porous dialysis membrane, said nanoparticles comprising at least one chelating agent as an active ingredient. include. In one embodiment, the cutoff threshold of the porous dialysis membrane is less than the mass of the chelating agent, ie, the mass of the nanoparticles comprising at least one chelating agent.

Alternatively, the perfusate is a colloidal suspension of a polymer whose average diameter is larger than the pores of the dialysis membrane, said polymer being grafted to at least one chelating active ingredient. In this regard, the cut-off threshold of the porous dialysis membrane is less than the mass of the chelating agent, ie the mass of the polymer to which at least one chelating agent is grafted.

According to the present invention, the term "colloidal suspension" means a mixture of a liquid and solid, insoluble particles that remain homogeneously dispersed, the particles being sufficient to keep the mixture stable and homogeneous. Often small (microscopic and submicroscopic).

According to one embodiment, the average diameter is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% larger than the pores of the dialysis membrane. .

According to the present invention, the term "average diameter" means the harmonic mean of the diameters of the nanoparticles or polymers to which at least one chelating agent is grafted. The size distribution of the nanoparticles or polymers can be measured, for example, using commercially available particle size analyzers, such as the Malvern Zeta Sizer Nano-S particle size analyzer based on photon correlation spectroscopy (PCS) characterized by mean hydrodynamic diameter. Ru. A method for measuring this parameter is also described in ISO 13321:1996.

In one embodiment, the colloidal suspension contains more than 1% by weight, in particular 2% by weight, 3% by weight, 4% by weight, 5% by weight, 6% by weight, 7% by weight, 8% by weight, 9% by weight %, preferably more than 10% by weight of nanoparticles or polymers.

Nanoparticles that can be used in devices according to the invention, especially dialysis systems or implants Nanoparticles that can be used in the invention have two basic characteristics.
- These are polysiloxane-based or silica-based.
- They have an average diameter of more than 3 nm, preferably less than 50 nm.

In one embodiment, said nanoparticles contain as an active ingredient at least one chelating agent capable of complexing with a metal cation, said chelating agent being greater than or equal to 10, preferably greater than or equal to 15. , has a complexation constant log(K _C1 ) with at least one of the metal cations.

According to the invention, the term "silica-based nanoparticles" means nanoparticles characterized by a mass percentage of silica of at least 8%.

According to the invention, the term "polysiloxane-based nanoparticles" means nanoparticles characterized by a mass percentage of silicon of at least 8%.

According to the invention, the term "polysiloxane" means an inorganic crosslinked polymer consisting of siloxane chains.

The structural units of polysiloxane, whether the same or different, have the following formula Si(OSi) _n R _4-n
has, in the formula,
- R is an organic molecule linked to silicon by a Si-C covalent bond;
- n is an integer from 1 to 4;

As a preferred example, the term "polysiloxane" in particular includes polymers resulting from the condensation of tetraethylorthosilicate (TEOS) and aminopropyltriethoxysilane (APTES) by a sol-gel process.

Advantageously, therefore, said nanoparticles: a. a polysiloxane in which the mass proportion of silicon is at least 8% of the total mass of the nanoparticles, preferably from 8% to 50% of the total mass of the nanoparticles;
b. chelating agent, preferably in a proportion of 5 to 1000, preferably 5 to 100, per nanoparticle;
c. If necessary, for example, a proportion of 5 to 100, preferably 5 to 20, metal elements per nanoparticle complexed to a chelating agent is included.

Even more advantageously, said nanoparticles have the following formula (I):
_Sin [O] _m [OH] _o [Ch ₁ ] _a [Ch ₂ ] _b [Ch ₃ ] _c [M ^y+ ] _d [D ^z+ ] _e [Gf] _f (I)
has
During the ceremony,
- n is 20 to 50,000, preferably 50 to 1000,
・m is greater than n and less than 4n,
・ o is 0 to 2n,
- Ch ₁ , Ch ₂ and Ch ₃ are the same or different and are chelating agents linked to the Si of the polysiloxane by a Si-C covalent bond, a, b and c are integers from 0 to n; , a+b+c is less than or equal to n, preferably a+b+c is between 5 and 100, such as between 5 and 20;
- M ^y+ and D ^z+ are the same or different metal cations, y and z are 1 to 6, d and e are integers from 0 to a+b+c, and d+e is less than or equal to a+b+c,
- Gf can be the same or different target grafts, each linked to Si by a Si-C bond, resulting from the grafting of the target molecule, thereby directing the nanoparticle to the desired biological tissue, e.g. tumor tissue. Targeting is enabled, and f is an integer from 0 to n.

In one embodiment, the nanoparticles usable according to the invention do not contain metallic elements. In other words, in the above definition said nanoparticles include a. (polysiloxane or silica) and b. Contains only (chelating agent).

In one embodiment, the chelating agent is complexed with cations of the metals Cu, Fe, Zn, Hg, Cd, Pb, Mn, Al, Ca, Mg, Gd.

In one embodiment, the chelating agent is a conjugating molecule or derivative thereof, such as especially DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N' ''-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DO3A-pyridine of the following formula (I),

EDTA (2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid), EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N', N'-tetraacetic acid), BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1 , 4,7-triacetic acid), DOTAGA ((2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanedionic acid), DFO (deferoxamine), for example DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) or NOTAM (1,4,7-tetrakis(carbamoylmethyl)-1 , 4,7-triazacyclononane) and mixed carboxylic acid/amide derivatives such as DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)) ) or phosphonic acid derivatives such as NOTP (1,4,7-tetrakis(methylenephosphonate)-1,4,7-triazacyclononane), TETA (1,4,8,11-tetraazacyclotetradecane-N, N',N'',N'''-tetraacetic acid), TETAM (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetrakis(carbamoylmethyl) ), cyclam derivatives such as TETP (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetrakis(methylenephosphonate)), or mixtures thereof. It is obtained by grafting (covalently bonding) polycarboxylic acids and one of their derivatives onto nanoparticles.

Preferably, the chelating agent is directly or indirectly covalently linked to the silica of the nanoparticle polysiloxane. The term "indirect" linkage refers to the presence of a molecular "linker" or "spacer" between the nanoparticle and the chelating agent, said linker or spacer being covalently bonded to one of the components of the nanoparticle. .

According to a preferred embodiment, the nanoparticles are polysiloxane-based nanoparticles having an average diameter of 3 to 50 nm and comprising a chelating agent obtained by grafting DOTA, DOTAGA or DTPA onto the nanoparticles. .

According to a preferred embodiment, said nanoparticles have an average dimension of more than 20 kDa and less than 1 MDa, and are polysiloxane-based nanoparticles comprising said chelating agent obtained by grafting DOTA, DOTAGA or DTPA onto the nanoparticles. It is a particle.

In a preferred embodiment, the colloidal suspension containing the nanoparticles also contains trace elements selected from calcium, magnesium, iron, copper, zinc or manganese.

Nanoparticles according to the invention can be obtained by the process described in patent application FR1053389.

Polymers that can be used in devices according to the invention In another embodiment of the invention, polymers can be used in place of the nanoparticles described above. In such cases, the polymer is grafted to at least one chelating agent.

According to the present invention, the term "polymer" means any macromolecule formed by a covalent arrangement of a large number of repeating units derived from one or more monomers. Polymers preferably used in the present invention are, for example, from the chitosan, polyacrylamide, polyamine or polycarboxylic acid families. For example, these may be polymers containing amino functional groups such as chitosan. According to a preferred embodiment, said polymer is biocompatible.

According to one embodiment, the chelating agent or derivative thereof grafted to said polymer is in particular DOTA, DTPA, DO3A-pyridine of formula (I) above, EDTA, EGTA, BAPTA, NOTA, DOTAGA, DFO, Polyaminopolycarboxylic acids and derivatives thereof selected from DOTAM, NOTAM, DOTP, NOTP, TETA, TETAM, and TETP, or mixtures thereof.

Preferably, the chelating agent described above is directly or indirectly covalently linked to the polymer or to a polymer chain of greater than 10 kDa, preferably greater than 100 kDa. The term "indirect" linkage refers to the presence of a molecular "linker" or "spacer" between the polymer and the chelating agent, said linker or spacer being covalently bonded to one of the components of said polymer.

In one embodiment, the chelating agent or derivative thereof grafted to the polymer will include a dithiocarbamate functionality.

According to a preferred embodiment, said polymer grafted with a chelating agent is selected from chitosan grafted with DPTA-BA or chitosan grafted with DFO.

In a preferred embodiment, the colloidal suspension containing the polymer also contains trace elements selected from calcium, magnesium, iron, copper, zinc or manganese.

Chelating molecules that can be used in devices according to the invention Alternatively, the perfusate is a solution of chelating molecules. The chelating molecules have an average diameter larger than the pores of the dialysis membrane, i.e. larger than the cut-off threshold of the membrane to be retained in the dialysis membrane fluid, or have an average diameter smaller than the pores of the porous dialysis membrane. diameter, in which case they enter the body after passing through the membrane pores and are naturally excreted by the kidneys or liver.

In this embodiment, said chelating molecule has a complexation constant log(K _C1 ) with at least one of said metal cations greater than or equal to 10, preferably greater than or equal to 15.

In this embodiment, the solution of chelating molecules also includes trace elements selected from calcium, magnesium, iron, copper, zinc, or manganese.

Chelating agent graft implants that can be used in devices according to the invention Alternatively, the means for extracting metal cations is an implant comprising at least one chelating agent.

According to one embodiment, the means for extracting metal cations is an implant onto which at least one chelating agent is grafted.

According to the invention, "implant" means any element intended to be introduced into the body. This may be a "polymer" or "any other solid" as described herein.

In this embodiment, polymers such as those described above can be used in the perfusate.

According to the present invention, the term "any other solid" is non-limiting and may optionally be surface-functionalized or non-surface-functionalized and may be of various shapes (spherical, tubular, flat means a ceramic, metal, composite, solid or porous part that may have a shape, etc.).

In this embodiment, the implant can be particularly temporarily implanted and then removed.

Preferentially, the implant can be implanted in the brain, liver, pancreas, etc. of the subject to be prevented and/or treated. The implant is resorbable and can be naturally and gradually excreted by the body. The implant may contain at least one chelating agent that slowly diffuses in the body, e.g. less than 100 mg of chelated molecules are released per day, preferably less than 10 mg per day, and/or per day. Diffusion of less than 1% of the total mass is possible. The implant may be placed in direct contact with tissue or subcutaneously.

Alternatively, the implant may be in a reservoir with dialysate in contact with the subject to be treated.

In a second aspect, the invention relates to the use of a colloidal suspension as described above, in particular in a device as described above.

The present invention therefore provides a colloidal suspension of nanoparticles containing an active ingredient for therapeutic use, comprising a device for maintaining the homeostasis of a metal comprising a porous dialysis membrane, said nanoparticles The present invention relates to a colloidal suspension characterized in that the average diameter of the pores of the porous dialysis membrane of the device is larger than the pores of the porous dialysis membrane of the device. Advantageously, said device is a microdialysis device.

In one embodiment, the present invention provides a colloidal suspension of a polymer grafted to an active ingredient for therapeutic use in a device for maintaining metal homeostasis, including a porous dialysis membrane. colloidal suspension, characterized in that the average diameter thereof is larger than the pores of the porous dialysis membrane, and that the polymer is grafted to the active ingredient.

Advantageously, said device is a microdialysis device.

In one embodiment, the invention provides that the device is capable of transporting it into a biological fluid such as blood, cerebrospinal fluid, synovial fluid, or ascites, or an organ such as the brain, liver, pancreas, intestine, or lungs. , or - any one of claims 1 to 13, characterized in that it comprises means allowing it to be placed in contact with or implanted in tissues such as the peritoneum or tumor tissue via a dialysis membrane. 1. A device for maintaining homeostasis of a metal according to item 1.

According to a preferred embodiment, the present invention relates to a colloidal suspension as described above for use in maintaining metal homeostasis.

According to another preferred embodiment, the invention relates to Parkinson's disease, Alzheimer's disease, neurodegeneration with brain iron accumulation (NBIA, also referred to as neurodegeneration with brain iron overload), Wilson's disease. or to a colloidal suspension as described above for use in the treatment of neurological diseases such as Huntington's disease or brain degeneration.

According to another preferred embodiment, the invention relates to a colloidal suspension as described above for use in the treatment of autism.

According to another preferred embodiment, the invention relates to a colloidal suspension as described above for use in the treatment of type II diabetes or cardiovascular diseases.

According to another preferred embodiment, the invention relates to a colloidal suspension as described above for use in the treatment of tumors.

In a third aspect, the invention relates to the use of nanoparticles as described above, in particular in devices as described above.

In one embodiment, the present invention therefore relates to polysiloxane-based nanoparticles having a diameter of more than 3 nm, preferably less than 50 nm, for therapeutic use in devices for maintaining homeostasis of metals, said nanoparticles having a diameter of more than 3 nm, preferably less than 50 nm. It contains as an active ingredient at least one chelating agent capable of complexing with a metal cation, and has a complexation constant log (K _C1 ) with at least one of said metal cations greater than 10, preferably greater than 15 or more. is characterized by being equal to . Advantageously, said device is a microdialysis device.

According to a preferred embodiment, the invention relates to nanoparticles as described above for use in maintaining homeostasis of metals.

In another preferred embodiment, the invention relates to nanoparticles as described above for use in the treatment of neurological diseases or brain degeneration, such as NBIA-type diseases, Parkinson's disease, Alzheimer's disease, Wilson's disease or Huntington's disease.

In another preferred embodiment, the invention relates to nanoparticles as described above for use in the treatment of autism.

In another preferred embodiment, the invention relates to nanoparticles as described above for use in the treatment of type II diabetes or cardiovascular diseases.

According to another preferred embodiment, the invention relates to nanoparticles as described above for use in the treatment of tumors.

In a fourth aspect, the invention relates to the use of a polymer as described above, in particular in a device as described above.

In one embodiment, the present invention therefore relates to a polymer for therapeutic use in a device for maintaining metal homeostasis, said polymer comprising at least one chelating agent capable of complexing with metal cations. grafted, characterized in that the complexation constant log (K _C1 ) with at least one of said metal cations is greater than or equal to 10, preferably greater than or equal to 15. Advantageously, said device is a microdialysis device.

According to a preferred embodiment, the invention relates to a polymer as described above for use in maintaining metal and/or protein homeostasis.

According to another preferred embodiment, the present invention relates to a polymer as described above for use in the treatment of neurological diseases or brain degeneration, such as diseases of the NBIA type, Parkinson's disease, Alzheimer's disease, Wilson's disease or Huntington's disease.

According to another preferred embodiment, the invention relates to a polymer as described above for use in the treatment of autism.

According to another preferred embodiment, the invention relates to the above-mentioned polymers for use in the treatment of type II diabetes or cardiovascular diseases.

According to another preferred embodiment, the invention relates to a polymer as described above for use in the treatment of tumors.

The present invention also provides methods for removing metal cations from a subject, including the administration of an implant having at least one chelating agent grafted thereon, or the use of a perfusate containing at least one chelating agent in a device as described above. Concerning a method for extracting.

According to the present invention, said "subject" means a human or animal to whom prophylaxis or treatment is provided.

The invention is best explained by the following examples and drawings. The following examples are intended to clarify the subject matter of the invention and to illustrate advantageous embodiments, but are not intended to limit the scope of the invention in any way.

FIG. 3 shows images obtained at the end of perfusion of _MnCl2 solution. This is a coronal section of the microdialysis membrane (black dots). The enhancement around the membrane corresponds to the presence of Mn ²⁺ (positive MRI contrast agent). FIG. 3 shows an image obtained at the end of perfusion with nanoparticle suspension. This is a coronal section of the microdialysis membrane (black dots). The enhancement around the membrane corresponds to the presence of Mn ²⁺ (positive MRI contrast agent). FIG. 2 shows an image that corresponds to the difference between the previous two images (shown in FIGS. 1 and 2) and highlights the decrease in the tissue concentration of Mn ²⁺ (highlighted in the microdialysis probe). FIG. 3 shows images obtained at the end of perfusion with _MnCl2 solution. This is a coronal section of the microdialysis membrane (black dots). The enhancement around the membrane corresponds to the presence of Mn ²⁺ (positive MRI contrast agent). FIG. 3 shows an image obtained at the end of saline perfusion. This is a coronal section of the microdialysis membrane (black dots). The enhancement around the membrane corresponds to the presence of Mn ²⁺ (positive MRI contrast agent). FIG. 5 shows an image corresponding to the difference between the previous two images (shown in FIGS. 4 and 5) and highlighting the absence of a decrease in the tissue concentration of Mn ²⁺ (little emphasis in the microdialysis probe). be. FIG. 3 shows MRI images of solutions 1, 2, 3, 4, and 5. FIG. 7 is a diagram showing the hydraulic diameter of nanoparticles obtained in Example 7. FIG. 7 is a diagram showing the hydraulic diameter of nanoparticles obtained in Example 8.

[Example]
(Example 1)
Extraction of Manganese Ions from Rodent Brains This study was carried out on male Wistar rats (body weight 250 g).

On day 0, the animals were placed under gas anesthesia (2.5% O ₂ /N ₂ (80:20)) using the heating mat used during the procedure and recovery phase for the insertion of the microdialysis cannula. isoflurane). Before incising the skin and cleaning the skull, perform local anesthesia with a subcutaneous injection of lidocaine (Xylovet 21.33 mg/mL) (4 mg/kg diluted in 0.9% NaCl, injection volume 10 μL/g). ). After the skin incision, the skull is cleaned in order to position a microdrill (less than 1 mm in diameter) for cranial puncture. The probe is inserted using stereotactic neurosurgery. A dialysis cannula (less than 500 μm in diameter) is gently inserted into the brain at the desired location and depth. After positioning the cannula, apply quick-curing fixation resin and screw it into the animal's skull. The skin is then sutured to close the wound. Analgesics (Buprecare) are administered subcutaneously before the animals wake up. Analgesic administration is repeated at 8-12 hour intervals for 2 days after inserting the microdialysis probe. To limit dehydration of the animals, a subcutaneous injection of 0.9% NaCl (approximately 0.5 mL for mice and 5 mL for rats) is given at the beginning of the procedure. Apply ophthalmic ointment (Liposic) at the beginning of the procedure to prevent dry eyes.

MRI spectroscopy and imaging protocols are performed on day 3. Under gas anesthesia (2.5% isoflurane under O ₂ /N ₂ (80:20)) with a heating mat used during the procedure and recovery phase and with respiratory control during NMR acquisition. The protocol is carried out on several animals. Insert the microdialysis probe (membrane length 2 mm, cutoff 6 kDa, CMA Microdialysis AB, Kista, Sweden) into the microdialysis cannula before positioning the animal during MRI (Bruker, Biospin, 4.7 Tesla). do. An MRI surface antenna (Doty Scientific, 8 mm diameter, used for transmitting and receiving) is positioned on the animal's skull perpendicular to the microdialysis probe. MRI acquisitions (T1 weighted flash sequence, echo time 2 ms, repetition time 150 ms, coronal sections, section thickness 1 mm, acquisition time 3 minutes) are performed continuously during microdialysis probe perfusion.

Results (Example 1A)
Perfuse the microdialysis probe with a 1 mM MnCl ₂ solution in saline for 30 min at a flow rate of 10 μL/min. A suspension of polysiloxane nanoparticles (28.1 mg) with free DOTAGA on its surface was then diluted and equilibrated with 1 mL of saline and 100 μL of NaOH and HCl to a pH of 7, i.e. 28.1 mg in a total volume of 1100 μL. Perfuse the microdialysis probe with 1 mg) for 30 minutes at a flow rate of 10 μL/min. The polysiloxane nanoparticles used consist of a polysiloxane matrix grafted with a DOTAGA cyclic chelating agent. These nanoparticles have a hydrodynamic diameter of 11.5±6.3 nm. This dimension prevents these nanoparticles from passing through dialysis membranes with pore sizes of 2-3 nm.

The images obtained at the end of the perfusion with the _MnCl2 solution are shown in Fig. 1, and the images obtained at the end of the perfusion with the nanoparticle suspension are shown in Fig. 2. FIG. 3 shows an image that corresponds to the difference between the previous two images and highlights the decrease in tissue Mn ²⁺ concentration (highlighted in the microdialysis probe).

(Example 1B)
Perfuse the microdialysis probe with a 1 mM MnCl ₂ solution in saline for 30 min at a flow rate of 10 μL/min. The microdialysis probe is then perfused with saline for 30 minutes at 10 μL/min. The images obtained at the end of the perfusion with _MnCl2 solution are shown in Fig. 4, and the images obtained at the end of the perfusion with saline solution are shown in Fig. 5. FIG. 6 shows an image that corresponds to the difference between the previous two images and shows that there is no decrease in the tissue concentration of Mn ²⁺ (little enhancement in the microdialysis probe).

Conclusion MRI allows for the demonstration of variations in tissue concentrations of Mn ²⁺ cations (paramagnetic MRI contrast agents). The presence of chelated nanoparticles in the perfusate results in a significant decrease in intensity in MRI sections due to a decrease in local tissue Mn ²⁺ concentration. This decrease in intensity is not observed in the absence of chelating nanoparticles.

(Example 2)
Extraction of intratissue gadolinium by perfusion of nanoparticle solution This study was performed on male Wistar rats (body weight 250 g).

On day 0, the animals were placed under gas anesthesia (2.5% O ₂ /N ₂ (80:20)) using the heating mat used during the procedure and recovery phase for the insertion of the microdialysis cannula. isoflurane). Before incising the skin and cleaning the skull, perform local anesthesia with a subcutaneous injection of lidocaine (Xylovet 21.33 mg/mL) (4 mg/kg diluted in 0.9% NaCl, injection volume 10 μL/g). ). After the skin incision, the skull is cleaned in order to position a microdrill (less than 1 mm in diameter) for cranial puncture. The probe is inserted using stereotactic neurosurgery. A dialysis cannula (less than 500 μm in diameter) is gently inserted into the brain at the desired location and depth. After positioning the cannula, apply quick-curing fixation resin and screw it into the animal's skull. The skin is then sutured to close the wound. Analgesics (Buprecare) are administered subcutaneously before the animals wake up. Analgesic administration is repeated at 8-12 hour intervals for 2 days after inserting the microdialysis probe. To limit dehydration of the animals, a subcutaneous injection of 0.9% NaCl (approximately 0.5 mL for mice and 5 mL for rats) is given at the beginning of the procedure. Apply ophthalmic ointment (Liposic) at the beginning of the procedure to prevent dry eyes.

A microdialysis perfusion protocol is performed on day 3. The protocol was performed on animals under gas anesthesia (2.5% isoflurane under O ₂ /N ₂ (80:20)), with heating mats used during the procedure and recovery phase, and with controlled breathing frequency. implement. A microdialysis probe (membrane length 2 mm, cutoff 6 kDa, CMA Microdialysis AB, Kista, Sweden) is inserted into the microdialysis cannula and perfusion is performed at a flow rate of 10 μL/min.

Perfusion is carried out for 30 minutes using a perfusion solution consisting of saline supplemented with 1 mM _GdCl (solution 1). Collect dialysate at the end of microdialysis (solution 2).

Nanoparticle suspension VL29-5 (28.1 mg was diluted and equilibrated with 1 mL of saline and 100 μL of NaOH and HCl to pH 7, i.e. 28.1 mg in a total volume of 1100 μL) for 30 min. , perfuse the microdialysis probe (solution 3). Collect dialysate at the end of microdialysis (solution 4). The nanoparticles used are the same as in Example 1. That is, they have a hydraulic diameter of 11.5±6.3 nm. This dimension prevents these nanoparticles from passing through dialysis membranes with pore sizes of 2-3 nm.

These four solutions (as well as saline solution 5) are imaged with a 4.7 Tesla MRI using a T1 weighted gradient echo sequence (repetition time 40 ms, echo time 2.6 ms, tilt angle 80°).

Images of the five tubes are shown in Figure 7.

Results The results in Figure 7 thus highlight the increased strength of solution 4 compared to solution 5, which is due to the uptake and chelation of tissue Gd while the nanoparticle solution passes through the microdialysis probe. This clearly shows that transformation is occurring.

(Example 3)
Synthesis of Chitosan-DTPA-BA The chitosan used has an average molecular weight of 200 kDa. DTPA-BA (diethylenetriaminepentaacetic dianhydride was supplied by Chematech, Dijon, France and used as received. VIVAFLOW cassette was purchased from Sartorius and used as received. Perfusion fluid (Perfusion Fluid CNS Sterile, product no. P000151) was purchased from Phymep and used as received.

Chitosan having a mass of 0.5 g was weighed and placed in a 500 mL container. A volume of 250 mL of distilled water was added and the solution was stirred. The pH was adjusted to 4.0±0.1 using a pH meter and 50% acetic acid solution. The solution was stirred for 24 hours. After 24 hours, the pH was again adjusted to 4.0±0.1. This procedure was repeated until all chitosan was completely dissolved.

DTPA-BA having a mass of 5.36 g was weighed and added to the resulting solution. The solution was stirred for 48 hours. After 48 hours, the solution was purified using a Vivaflow cassette with a cutoff of 100 kDa to a purity of at least 100,000-fold. Replace the solvent with CNS perfusate at the same concentration using the Vivaflow cassette again.

(Example 4)
Synthesis of Chitosan-DFO The chitosan used has an average molecular weight of 200 kDa. p-NCS-Bz-DFO(N1-hydroxy-N1-(5-(4-(hydroxy(5-(3-(4-isothiocyanatophenyl)thiourido)pentyl)amino)-4-oxobutanamido)pentyl )-N4-(5-(N-hydroxyacetamido)pentyl)succinamide) was purchased from Chematech Mdt and used as received. VIVAFLOW cassettes were purchased from Sartorius and used as received. Perfusion fluid (Perfusion Fluid CNS Sterile, product number P000151) was purchased from Phymep and used as received.

Chitosan having a mass of 0.5 g was weighed and placed in a 500 mL container. A volume of 250 mL of distilled water was added and the solution was stirred. The pH was adjusted to 4.0±0.1 using a pH meter and 50% acetic acid solution. The solution was stirred for 24 hours. After 24 hours, the pH was again adjusted to 4.0±0.1. This procedure was repeated until all chitosan was completely dissolved.

A mass of 500 mg of p-NCS-Bz-DFO was weighed and added to the resulting solution. The solution was stirred for 48 hours. After 48 hours, the solution was purified using a 100 kDa cutoff Vivaflow cassette to reach a purification level of at least 100,000-fold. Replace the solvent with CNS perfusate at the same concentration using the Vivaflow cassette again.

(Example 5)
Purification of MetalSorb and Condition Adjustment Metalsorb FZ, a polyacrylamide polymer containing dithiocarbamate functional groups, was supplied by SNF, France and was used as received. VIVAFLOW cassettes were purchased from Sartorius and used as received. Perfusion fluid (Perfusion Fluid CNS Sterile, product number P000151) was purchased from Phymep and used as received.

A volume of 50 mL of Metalsorb 20% w/w was measured into a 250 mL container. A volume of 150 mL of water was added and the solution was stirred for 2 hours. After 2 hours, the solution was purified using a Vivaflow cassette with a cutoff of 100 kDa to a purity of at least 100,000-fold. The solvent was replaced with CNS perfusate at the same concentration using the Vivaflow cassette again.

(Example 6)
Use of the materials obtained in Examples 3, 4 and 5 The materials obtained in Examples 3, 4 and 5 above can be advantageously used as a means for extracting metal cations according to the invention. . The solution can be used directly or by adapting the formulation to form a perfusate, or the polymer may be extracted and solidified to form a macroscopic solid that can be implanted.

(Example 7)
Synthesis of Polysiloxane-EDTA Nanoparticles Polysiloxane particles containing the EDTA (ethylenediaminetetraacetic acid) type chelate Si@EDTA were synthesized using three silane precursors: (i) TEOS (tetraethylorthosilicate)-((Si(OC ₂ H ₅ ) ₄ , 98%-Sigma Aldrich Chemicals, France)), (ii) APTES (3-(3-aminopropyl)triethoxysilane-(H ₂ N(CH ₂ ) ₃ -Si(OC ₂ H ₅ ) ₃ , 99%-Sigma Aldrich Chemicals, France)), and (iii) Si-EDTA (N-(trimethoxysilylpropyl)ethylenediaminetriacetic acid trisodium salt-(N-[3-trimethoxysilylpropyl]ethylenediamine trisodium salt)). DEG (diethylene glycol-DEG) in a molar ratio of 2:1:3 (TEOS/APTES/Si-EDTA). , 99%-SDS Carlo Erba (France).The mixture is kept under stirring at room temperature for 30 minutes, then 3 volumes of water are added and stirred again at the same temperature for 17 hours.Then the temperature is lowered. Increase the temperature to 80 °C and maintain stirring for 6 hours (adjust the pH to a value of 7.4 after 2 hours of heating).Heating is then stopped and the solution is kept under stirring for 17 hours.The solution is then tangential flow filtered. The nanoparticles have a hydrodynamic diameter of 21±9 nm by dynamic light scattering (DLS) using a PCS-based Malvern Zeta Sizer Nano-S particle size analyzer (FIG. 8).

(Example 8)
Synthesis of Polysiloxane-DTPA Nanoparticles For nanoparticles containing chelates of the DTPA (diethylenetriaminepentaacetic acid) type, a preliminary step of grafting the chelate onto the silane is required. Silane containing DTPA was obtained by reacting a derivative of DTPA, DTPA-BA (diethylenetriaminepentaacetic dianhydride - CheMatech, Dijon, France) with APTES in DEG at a 1:1 ratio of DTPA-BA/APTES. It will be done. The solution is left under stirring for 24 hours. TEOS is then added in a TEOS/APTES/DTPA-BA ratio of 3:1:1. After stirring for 1 hour in DEG, water is added (10 times the volume of DEG used). The solution is then stirred at room temperature for 24 hours, heated to 50° C. and stirred again for 24 hours. Finally, the solution is cooled to room temperature and left under stirring for 72 hours. The nanoparticles are then purified by tangential flow filtration and the pH is raised to 7.4. The nanoparticles have a hydrodynamic diameter of 7±3 nm with a second population of 20±7 nm in DLS evaluated using a PCS-based Malvern Zeta Sizer Nano-S particle size analyzer (Figure 9).

(Example 9)
Comparison of Microdialysis Flow Rates in Extraction of Metals In this example, the extraction performance of a perfusate containing a chelating agent in a microdialysis device from an aqueous solution containing several metal ions was compared.

Several flow rates (1, 2, and 5 μL/min) were tested using the same perfusate (polysiloxane-EDTA nanoparticles whose synthesis was described in Example 8 with a concentration of 15 mM EDTA dispersed in water). . The microdialysis membrane (63 Microdialysis Catheter, M Dialysis AB, Sweden) had a cutoff of 20 kDa. The solution used to test the chelating ability of the perfusate was an aqueous solution containing Al(III), Cd(II), Zn(II), Cu(II), and Pb(II) ions at a concentration of 100 ppb each. It was a solution. The pH of the solution was adjusted to 7.4 and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma Aldrich Chemicals, France) was added as a buffer at a concentration of 1.2 g/L. The total volume of the solution is 600 mL.

Extraction by microdialysis required 40 minutes at flow rates of 2 and 5 μL/min. A 1 μL/min sample was obtained in 100 minutes. These samples were analyzed by ICP/MS and the amount of each metal is reported in Table 2. This experiment was performed four times and for each of the metals tested under all conditions using a perfusate based on chelated nanoparticles, using conventional microdialysis where the perfusate initially contains only water ( _H2O ). shows better extraction compared to When using perfusate containing chelating agents, uptake of metals at concentrations exceeding their "diffusion concentration" in the medium to be purified was observed. Chelation is particularly effective in the case of aluminum because of its small dimensions, which allows faster diffusion through the membrane. A flow rate of 2 μL/min seemed to be a good compromise between efficient extraction and sample volume and was selected for Examples 10 and 11.

(Example 10)
Comparison of perfusion solutions based on polysiloxane-DTPA and polysiloxane-EDTA nanoparticles. Using a microdialysis membrane with a cutoff of 20 kDa, a microdialysis flow rate of 2 μL/min, and a sample collection time of 40 minutes, as described in Example 9. The relative efficiency of the nanoparticles obtained in Examples 7 and 8 was compared using the same metal mixture. Results were obtained using three different perfusion fluids, (i) water, (ii) polysiloxane-EDTA nanoparticles, and (iii) polysiloxane-DTPA nanoparticles, with a chelating agent concentration of 15 mM. The results are summarized in Table 3. DTPA-based nanoparticles have an extremely high affinity for aluminum as a chelating agent, and therefore have an extremely high extraction capacity for aluminum. It appears that the presence of aluminum saturates the surface chelators and reduces the efficiency of the perfusate towards other metals. Polysiloxane-DTPA nanoparticles make it possible to obtain a perfusate that is highly specific for the extraction of aluminum.

(Example 11)
Use of polysiloxane-EDTA nanoparticles as perfusion fluid for cerebrospinal fluid (CSF) To model CSF, NaCl (147 mM), KCl (2.7 mM), _CaCl2 (1.2 mM), and MgCl A solution containing ₂ (0.85mM) was synthesized. This solution was used to extract metals to ensure that the extraction power was not diminished by various potentially interfering ions. A solution similar to that in Example 9 (i.e., 600 mL of reconstituted CSF containing 100 ppb of each of Al(III), Cd(II), Zn(II), Cu(II), and Pb(II) ions) )It was created. The microdialysis membrane used (63 Microdialysis Catheter, M Dialysis AB, Sweden) had a cutoff of 20 kDa, the flow rate was set at 2 μL/min, and the collection time was 40 minutes. The amount of extracted metals was analyzed by ICP/MS. The perfusate consisted of either reconstituted CSF or polysiloxane-EDTA nanoparticles dispersed in reconstituted CSF, the synthesis of which is described in Example 7. The extraction results are shown in Table 4. It is noted that the extraction volume of perfusate containing only CSF is extremely low. By adding nanoparticles to the perfusate, metal extraction is significantly increased regardless of the metal. These conditions yield metal extraction factors greater than 5 for lead, greater than 7 for copper, greater than 25 for cadmium, and greater than 125 for aluminum.

Claims (24)

A device for maintaining metal homeostasis for therapeutic purposes, the device comprising:
a. a porous dialysis membrane, and b. a dialysis system including a reservoir containing perfusate;
The perfusate is
(1) A colloidal suspension of nanoparticles having an average diameter larger than the pores of the porous dialysis membrane, wherein the nanoparticles are conjugated with at least one chelating agent capable of complexing with metal cations. (2) of a polymer larger than 10 kDa covalently grafted directly or indirectly to at least one chelating agent capable of complexing with a metal cation . colloidal suspension ,
A device containing any of the following. characterized in that the chelating agent is capable of complexing with a metal cation, and characterized in that the complexing constant log(K _C1 ) of the chelating agent with at least one of the metal cations is greater than 10. A device for maintaining homeostasis of a metal according to claim 1. characterized in that said chelating agent is capable of complexing with metal cations, said chelating agent having a complexation constant log (K _C1 ) with at least one of said metal cations greater than or equal to 15; Device for maintaining homeostasis of metals according to claim 2, characterized in that: characterized in that said chelating agent is capable of complexing with metal cations, said cations being selected from cations of metals Cu, Fe, Zn, Hg, Cd, Pb, Mn, Mg, Ca, Gd, and Al; Device for maintaining homeostasis of metals according to claim 2, characterized in that: 5. Metal homeostasis according to claim 4, characterized in that said chelating agent is capable of complexing with metal cations, said cations being selected from cations of metals Cu, Fe and Zn. device for maintaining. Device for maintaining metal homeostasis according to any one of claims 1 to 5, characterized in that it comprises trace elements selected from calcium, magnesium, iron, copper, zinc or manganese. 7. Any of claims 1 to 6, characterized in that said means make it possible to extract said metal cations from biological fluids, organs or tissues when their content is less than 1 ppm. A device for maintaining homeostasis of a metal according to item 1. Device for maintaining metal homeostasis according to claim 7, characterized in that the content of metal cations is less than 0.1 ppm. Device for maintaining metal homeostasis according to claim 7, characterized in that the content of metal cations is less than 0.01 ppm. Device for maintaining metal homeostasis according to claim 7, characterized in that the content of metal cations is less than 1 ppb. Homeostasis of metals according to any one of claims 1 to 10, characterized in that the chelating agent makes it possible to extract metal cations in an amount corresponding to at least 1% of its mass. device to maintain. Device for maintaining homeostasis of metals according to claim 11, characterized in that the chelating agent makes it possible to extract metal cations in an amount corresponding to more than 10% of their mass. Device for maintaining metal homeostasis according to claim 1, characterized in that the colloidal suspension contains more than 1% by weight of nanoparticles or polymers. Device for maintaining metal homeostasis according to claim 1, characterized in that the colloidal suspension contains more than 10% by weight of nanoparticles or polymers. Device for maintaining homeostasis of metals according to any one of claims 1 to 7, characterized in that the nanoparticles are polysiloxane-based nanoparticles with an average diameter greater than 3 nm. Device for maintaining homeostasis of metals according to claim 15, characterized in that the nanoparticles are polysiloxane-based nanoparticles with an average diameter of less than 50 nm. The nanoparticles are
a. a polysiloxane in which the mass proportion of silicon is at least 8% of the total mass of said nanoparticles;
b. chelating agent at a rate of 5 to 1000 per nanoparticle;
c. Device for maintaining metal homeostasis according to any one of claims 1 to 8, characterized in that it comprises a proportion of 5 to 100 of the metal element complexed to the chelating agent. The nanoparticles are
a. a polysiloxane in which the mass ratio of silicon is 8% to 50% of the total mass of the nanoparticles;
b. chelating agent at a rate of 5 to 100 per nanoparticle;
c. Device for maintaining metal homeostasis according to claim 17, characterized in that it comprises a proportion of 5 to 20 metal elements complexed to the chelating agent per nanoparticle. The nanoparticles have the following formula (I)
_Sin [O] _m [OH] _o [Ch ₁ ] _a [Ch ₂ ] _b [Ch ₃ ] _c [M ^y+ ] _d [D ^z+ ] _e [Gf] _f (I)
has
During the ceremony,
n is 20 to 50,000,
m is greater than n and less than 4n,
o is 0 to 2n,
Ch ₁ , Ch ₂ and Ch ₃ are the same or different and are chelating agents linked to the Si of the polysiloxane by a Si-C covalent bond, a, b and c are integers from 0 to n; a+b+c is less than or equal to n;
M ^y+ and D ^z+ are the same or different metal cations, y and z are 1 to 6, d and e are integers from 0 to a+b+c, and d+e is less than or equal to a+b+c;
Gf can be the same or different targeting grafts, each linked to Si by a Si-C bond, resulting from the grafting of targeting molecules, thereby allowing targeting of the nanoparticles to the desired biological tissue. , f is an integer from 0 to n. Device for maintaining homeostasis of a metal according to any one of claims 1 to 9. The nanoparticles have the following formula (I)
_Sin [O] _m [OH] _o [Ch ₁ ] _a [Ch ₂ ] _b [Ch ₃ ] _c [M ^y+ ] _d [D ^z+ ] _e [Gf] _f (I)
has
During the ceremony,
n is 50 to 1000,
m is greater than n and less than 4n,
o is 0 to 2n,
Ch ₁ , Ch ₂ and Ch ₃ are the same or different and are chelating agents linked to Si of the polysiloxane by a Si-C covalent bond, a+b+c is 5 to 100,
M ^y+ and D ^z+ are the same or different metal cations, y and z are 1 to 6, d and e are integers from 0 to a+b+c, and d+e is less than or equal to a+b+c;
Gf can be the same or different target grafts, each linked to Si by a Si-C bond, resulting from the grafting of a targeting molecule, thereby allowing the targeting of nanoparticles to tumor tissue, where f is 0 Device for maintaining homeostasis of metals according to claim 19, characterized in that it is an integer of ~n. The chelating agent to which the nanoparticles have been grafted or the polymer has been grafted may be one of the following chelating agents capable of complexing with metal cations (DOTA, DTPA, EDTA, EGTA, BAPTA, NOTA, DOTAGA, DFO, DOTAM, NOTAM, DOTP, NOTP, TETA, TETAM, TETP, and DTPABA, or mixtures thereof ) according to any one of claims 1 to 20, characterized in that Device for maintaining homeostasis of the described metals. 22. According to any one of claims 1 to 21, characterized in that the nanoparticles are polysiloxane-based nanoparticles with an average diameter of 3 to 50 nm, and the chelating agent is DOTA, DOTAGA or DTPA. Device for maintaining homeostasis of the described metals. 23. Any one of claims 1 to 22, characterized in that the nanoparticles are polysiloxane-based nanoparticles with an average size greater than 20 kDa and less than 1 MDa, and the chelating agent is DOTA, DOTAGA, or DTPA. A device for maintaining homeostasis of metals as described in Section. The device,
Biological fluids such as blood, cerebrospinal fluid, synovial fluid, or ascites; or organs such as the brain, liver, pancreas, intestines, or lungs; or tissues such as the peritoneum or tumor tissue through a dialysis membrane. 24. A device for maintaining the homeostasis of metals according to any one of claims 1 to 23, characterized in that it comprises means for making it possible to be embedded in, or embedded in, a metal.